The present invention relates to novel cationic amphiphiles containing novel N-hydroxyalkyl group and therapeutic formulation containing the said amphiphiles that are capable of facilitating transport of biologically active molecules into cells. The invention further provides a process for the preparation of the said thereapeutic formulation containing amphiphilic compounds. The novel cationic amphiphiles containing N-hydroxyalkyl group of this invention are potentially useful to deliver therapeutically effective amounts of biologically active molecules into the cells of mammals. The area of medical science that is likely to benefit most from the present invention is gene therapy.
In gene therapy, patients carrying identified defective genes are supplemented with the copies of the corresponding normal genes. However, genes (DNA), the polyanionic macromolecules and the cell surfaces of the biological membranes both being negatively charged, spontaneous entry of normal copies of genes into the target cells of patients is an inefficient process (because of electrostatic repulsion). This is why the past decade has witnessed an unprecedented upsurge of global interest in developing efficient gene delivery reagents for introducing normal genes into the target cells of patients suffering from various genetic (inherited) diseases such as cystic fibrosis, Gaucher""s illness, Fabry""s disease etc. Many gene delivery reagents (also known as transfection vectors) including retrovirus, adenovirus, and cationic amphiphilic compounds (i.e. compounds containing both polar and non-polar functionalities) are being used as the carriers of polyanionic genes in combating hereditary diseases in gene therapy. The amphiphilic nature (presence of both polar and non-polar regions in the molecular structures) of the compounds designed to deliver therapeutically actives molecules, ensures smooth interaction of these carrier molecules with the polar and non-polar regions of plasma membranes, compartments within the cells and the biologically active molecules itself. At physiological pH, the cationic amphiphiles in the form of liposomes or micelles associate favorably with the negatively charged regions of the macromolecular polyanionic DNA enhancing the intracelluar uptake of the resulting complex between the cationic lipids and the negatively charged DNA. Reproducibility, high degree of targetability and low cellular toxicity are increasingly making the cationic amphiphiles the transfection vectors of choice in gene therapy.
An impressive number of cationic lipids with varying structures have been reported for the intracellular delivery of therapeutically active molecules as exemplified by the following references:
Feigner et al., Proc. Natl. Acad. Sci. U.S.A., 84, 7413-7417 (1987), reported the first use of a highly efficient cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) as the DNA transfection vector.
U.S. Pat. Nos. 4,897,355 and 4,946,787 (1990) reported the synthesis and use of N- [.omega..(.omega.- 1)-dialkyloxy]- and N-[..omega..(.omega.-l1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium amphiphiles and their pharmaceutical formulation as efficient transfection vectors.
Leventis, R. and Silvius, J. R. Biochim. Biophys. Acta. 1023, 124-132, (1990) reported the interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles.
U.S. Pat. No. 5,264,618 (1993) reported the synthesis and use of additional series of highly efficient cationic lipids for intracellular delivery of biologically active molecules.
Feigner et al. J Biol. Chem. 269 , 2550-2561 (1994) reported enhanced gene delivery and mechanistic studies with a novel series of cationic lipid formulations.
U.S. Pat. No. 5,283,185 (1994) reported the synthesis and use of 3xcex2[N-(N1,N1-dimethylaminoethane)carbamoyl] cholesterol, termed as xe2x80x9cDC-Cholxe2x80x9d for delivery of a plasmid carrying a gene for chloramphenicol acetyl transferase into cultured mammalian cells.
U.S. Pat. No. 5,283,185 (1994) reported the use of N-[2-[(2,5-bis[(3-aminopropyl)amino]- 1-Oxopentyl)aminoethyl]-N,N-dimethyl-2,3-bis-(9-Octadecenyloxy)-1-Propanaminium tetra(trifluoroacetate), one of the most widely used cationic lipids in gene delivery. The pharmaceutical formulation containing this cationic lipid is sold commercially under the trade name xe2x80x9cLipofectaminexe2x80x9d.
Solodin et al. Biochemistry 34, 13537-13544, (1995) reported a novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery.
Wheeler et al. Proc. Natl. Acad.Sci. U.S.A. 93, 11454-11459, (1996) reported a novel cationic lipid that greatly enhances plasmid DNA delivery and expression in mouse lung.
U.S. Pat. No. 5,527,928 (1996) reported the synthesis and the use of N,N,N,N-tetramethyl-N,N-bis(hydroxy ethyl)-2,3-di(oleolyoxy)-1,4-butanediammonium iodide i.e. pharmaceutical formulation as transfection vector.
U.S. Pat. No. 5,698,721 (1997) reported the synthesis and use of alkyl O-phosphate esters of diacylphosphate compounds such as phosphatidylcholine or phosphatidylethanolamine for intracellular delivery of macromolecules.
U.S. Pat. Nos. 5,661,018; 5,686,620 and 5,688,958 (1997) disclosed a novel class of cationic phospholipids containing phosphotriester derivatives of phosphoglycerides and sphingolipids efficient in the lipofection of nucleic acids.
U.S. Pat. No. 5,614,503 (1997) reported the synthesis and use of an amphipathic transporter for delivery of nucleic acid into cells, comprising an essentially nontoxic, biodegradable cationic compound having a cationic polyamine head group capable of binding a nucleic acid and a cholesterol lipid tail capable of associating with a cellular membrane.
U.S. Pat. No. 5,705,693 (1998) disclosed the method of preparation and use of new cationic lipids and intermediates in their synthesis that are useful for transfecting nucleic acids or peptides into prokaryotic or eukaryotic cells. These lipids comprise one or two substituted arginine. lysine or ornithine residues. or derivatives thereof. linked to a lipophilic moiety.
U.S. Pat. No. 5,719,131 (1998) has reported the synthesis of a series of novel cationic amphiphiles that facilitate transport of genes into cells. The amphiphiles contain lipophilic groups derived from steroids. from mono or dialkylamines, alkylamines or polyalkylamines.
Although the above mentioned cationic lipids have been successfully exploited for the intracellular delivery of genes, the efficiencies for the intracellular uptake procedures are insufficient and need to be improved. The transfection activities of most of the above mentioned cationic lipids are modest and therefore substantial quantities of these cationic lipids must be consumed. The associated cellular toxicities of the lipids and the metabolites thereof are, thus naturally, issues of concern. Accordingly, demands for developing new class of cationic amphiphiles with high transfection efficiencies and low cellular toxicities continue in this field of art.
The main objective of the invention is to provide novel cationic amphiphilic compounds containing non-toxic N-hydroxyalkyl group.
Another objective of the invention is to provide novel cationic amphiphilic compounds, which are useful for delivery of therapeutically effective amounts of biologically active molecules into cells/tissues of mammals.
Yet another objective of the invention is to provide cationic amphiphilic compounds such that a hydrophobic group is either directly linked to the positively charged Nitrogen atom or is linked to the said Nitrogen atom via an ester or methylene group.
Still another objective of the invention is to provide novel cationic amphiphilic compounds with at least one hydroxyalkyl group containing 1-3 carbon atoms directly linked to the positively charged Nitrogen atom.
Yet another objective of the invention is to provide novel cationic amphiphilic compounds without any glycerol backbone in their structure.
Another objective of the invention is to provide novel therapeutic formulation comprising one or more of the cationic amphiphilic compounds of the invention.
It is a further objective of the invention to provide therapeutic formulation useful in gene therapy and delivery of biologically active molecules into cells/tissues of mammals.
It is yet another objective to provide for intravenous administration of the therapeutic formulation for targeting organs for gene therapy.
The present invention relates to novel cationic amphiphiles containing N-hydroxyalkyl group. The novel cationic amphiphiles containing N-hydroxyalkyl group of this invention are potentially useful to deliver into the cells of patients therapeutically effective amounts of biologically active molecules. The area of medical science that is likely to benefit most from the present invention is gene therapy.
Cationic amphiphiles disclosed in the present invention possess several novel structural features. These features may be seen in comparison with, for example, cationic amphiphilic structures such as those disclosed in Felgner et al. J. Biol. Chem., 269, 2550-2561 (1994), a representative structure of which is 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (xe2x80x9cDMRIExe2x80x9d) and to those disclosed by Bennett et al. J Med Chem., 40, 4069-4078 (1997), a representative structure of which is N,N-[Bis(2-hydroxyethyl)]-N-methyl-N-[2,3-bis(tetradecanoyloxy)propyl] ammonium chloride (xe2x80x9cDMDHPxe2x80x9d).
The following distinctive structural features are common to the cationic amphiphiles disclosed in the present invention: (1) the presence of a hydrophobic group which is either directly linked to the positively charged nitrogen atom or is linked to the positively charged nitrogen via an ester group. (2) the presence of at least one hydroxyalkyl group containing 1-3 carbon atom that is directly linked to the positively charged nitrogen atom and (3) unlike many other glycerol-based cationic amphiphiles, the cationic transfection lipids disclosed in the present invention do not have any glycerol backbone in their molecular architecture. It is believed that these unique structural features contribute significantly to the increased transfection efficiencies of the cationic amphiphiles disclosed herein. The enhanced in vitro transfection efficiencies of N,N,-di-[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N-methylammonium bromide (DOHEMAB) and N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium bromide (DHDEAB), the two novel transfection lipids of the present invention, is compared to Lipofectamine, the most widely used commercially available highly efficient transfection lipid.
According to the practice of the present invention, xe2x80x9ccationicxe2x80x9d means the positive charge is either on a quaternized nitrogen atom or on a protonated tertiary nitrogen atom. The cationic characters of the amphiphiles may contribute to the enhanced interaction of the amphiphiles with biologically active molecules (such as nucleic acids) and/or with cell constituents such as plasma membrane glycoproteins. Such enhanced interaction between the cationic transfection lipid and the biological by active molecules and/or cell membrane constituents may play a key role in successfully transporting the therapeutic molecules into the cells.
The cationic amphiphiles of the present invention have certain common structural and functional groups. As such, the said cationic amphiphilic compounds may be represented by the following generic formula: 
Wherein
n is an integer between 1 and 3.
R1 may be H. saturated or unsaturated aliphatic group (C8-C20) or long chain saturated or unsaturated alkyl group (C7-C19).
R3 may be hydroxyalkyl group of 1-3 carbon atoms.
R2 may be a long chain saturated alkyl group (C7-C19) or [(CH2)nxe2x80x94Zxe2x80x94R2]
Z may be methylene group (xe2x80x94CH2), or an esteric group (xe2x80x94Oxe2x80x94COxe2x80x94)
Various novel amphiphilic compounds having the above basic structural formula may be synthesised by the processes described in co-pending U.S. patent application Ser. No. 09/275,816 (Indian Application No. 3324/DEL/98, 3325/DEL/98 and 3327/DEL/98 all dated Nov. 9, 1998). The cationic amphiphiles disclosed in the present invention share certain common structural features as discussed herein below. Further, in the said amphihpiles the preference of C8-C20 is specifically low because below the chain length of C8 the amphiphiles loose the aggregation property and the specific reason for not using carbon chains longer than C20 is because they are not compatible with biological membranes in terms of their lengths. Further, the additional linking groups that may be practised within the scope of this invention include xe2x80x94Oxe2x80x94(ether) CONHxe2x80x94(amide) group etc. Among the three different linking groups described in the invention herein below, the esteric group is most preferred.
Further, the products obtained during the synthesis of the said amphiphilic compounds are hydrolysis products. The moleclues were so constructed to aid in their breakdown within a cell subsequent to performing the task of transfection. The structure of the amphiphiles may also be altered by a combination of alkyl and amine moieties, which structures would fall within the teachings and scope of the present invention. Such modifications are apparent to those skilled in the art. Accordingly, certain representative amphiphilic compounds that may be practiced within the scope of the invention are described hereinbelow, along with their respective structural formulae:
Accordingly, the invention provide cationic amphiphiles represented by the following structural formula (I): 
wherein:
n is an integer between 1 and 3;
R1 represents either H or a saturated aliphatic group;
Z represents a methylene (xe2x80x94CH2xe2x80x94) group;
R2, independently, represents a long-chain saturated alkyl group (from C7 to C19);
R3 is a small hydroxyalkyl group consisting of 1-3 carbon atoms;
X is either a halogen atom or a tosylate group.
In one preferred embodiment of the cationic lipids, when n is 2, R1 is selected from H or C8 to C20 saturated alkyl groups, R2 is selected from C7 to C19 saturated alkyl groups, R3 is hydroxyethyl group (xe2x80x94CH2xe2x80x94CH2xe2x80x94OH) and X is selected from Br and Cl.
In one preferred embodiment n=2; Z=CH2; R3 is 2-hydroxylethyl group; R1 is selected from the n-C14H39 to n-C20H41. R2 is selected from n-C11H23 to n-C17H35 and X is selected from Br or Cl. According to this aspect of invention, particularly effective amphiphile includes, for example, N,N-di-n-hexadecyl-N,N-dthydroxyethylammonium bromide (DHDEAB, amphiphile No. 1).
In another preferred embodiment, n=2; Z=CH2; R3 is 2-hydroxyethyl group; R1 is selected from the n-C14H39 to n-C20H41. R2 is selected from the n-C11H23 to n-C17H35 and X is selected from Br or Cl. According to this aspect of invention, particularly effective amphiphile would include, for example, NN-di-n-hexadecyl-N,N dthydroxyethylammonium bromide (DHDEAB, amphiphile No. 1).
In another preferred embodiment, n=2; Z=CH2; R3 is 2-hydroxyethyl group; R1 is H and R2 is selected from the n-C11H23 to n-C17H35. According to this aspect of invention, particularly effective amphiphile would include, for example, N-n-hexadecyl-N, N dihydroxyethylammonium bromide (HDEAB, amphiphile No. (2).
The said pair of particularly effective representative cationic amphiphiles N,N-di-n-hexadecyl-N, N-dihydroxyethylammonium bromide (DHDEAB), amphiphile No 1 and N-n-hexadecyl-N,N-dihydroxyethylammonium bromide (HDEAB), amphiphile No. 2 are represented by the following structural formulae: 
Accordingly the invention provides cationic amphiphiles having, structural formula (II) as represented hereunder: 
wherein:
n is an integer between 1 and 3;
R1 independently, represents either a saturated aliphatic group or an unsaturated aliphatic group (from C8 to C20): Z represents a methylene (xe2x80x94CH2xe2x80x94) group:
R2 independently represents a long-chain saturated alkyl group (from C7 to C19);
R3 is a small alkyl group (from C1 to C3);
X is either a halogen atom or a tosylate group.
In one preferred embodiment, when n is 2, R1 is selected independently from C8 to C20 saturated or unsaturated alkyl groups, R2 is selected from C7 to C19 saturated alkyl groups, R3 is selected from small alkyl groups (from C1 to C3) and X is selected from Br and Cl.
In a preferred embodiment, n=2; Z=CH2, R3 is selected from methyl or ethyl group; R1 is selected from the C14H27 to C20H39, R2 is selected from n-C12H23 to n-C17H35 and X is selected from Br or Cl. According to this aspect of invention, particularly effective amphiphile includes, for example, N-methyl-N-n-octadecyl-N-oleyl-N-hydroxyethyl-ammonium chloride (MOOHAC, amphiphile No. 3)
In a further preferred embodiment, n=2, Z=CH2 R3 is selected from methyl or ethyl group; R1 is selected from the n-C1H29 to n-C20H41, R2 is selected from n-C1H23 to n-C12H35 and X is selected from Br of Cl. According to this aspect of invention, particularly effective amphiphile includes, for example, N,N-di-n-octadecyl-N-methyl-N-dihydroxyethylammonium chloride (DOMHAC, amphiphile No. 4)
According to an embodiment, an effective representative pair of cationic transfection lipids include, for example, N-methyl-N-n-octadecyl-N-oleyl-N-hydroxyethyl-ammonium chloride (MOOHAC), amphiphile No 3 and N,N-di-n-octadecyl-N-methyl-N-dihydroxyethylammonium chloride (DOMHAC). amphiphile No. 4 represented by their structural formulae as under: 
Additionally, the present invention provides novel cationic amphiphiles that facilitate the intracellular delivery of biologically active molecules, said amphiphiles having the structural formula (III). 
wherein:
n is an integer between 1 and 3, Z an ester group (xe2x80x94Oxe2x80x94COxe2x80x94) and R1 and R2 independently, represent a long-chain saturated or unsaturated alkyl group (from C7 to C19), R3 can be either a small alkyl group (C1 to C3) and X is either a halogen atom or a tosylate group,
In an embodiment of the invention, n=2; Z=Oxe2x80x94COxe2x80x94; R3 is selected from methyl or ethyl group; R1 is selected from the n-C14H29 to n-C20H41, R2 is selected from n-C14H29 to n-C20H41 and X is selected from Br or Cl. According to this aspect of invention, particularly effective amphiphile includes, for example, NN-di(O-hexadecanoyl/hydroxyethyl-N-hydroxyethyl-N-methylammonium bromide (DOHEMAB, amphiphile No. 5).
According to this aspect of the invention, a particularly effective representative cationic transfection lipid includes N,N,-di[O-hexadecanoyl]hydroxyethyl-N-hydroxyethyl-N-methylammonium bromide (DOHEMAB), amphiphile 5 having the structural formula: 
Formulations
The invention also provides novel therapeutic formulation comprising therapeutically effective amounts of the cationic amphiphilic compounds disclosed in the present invention, biologically active molecules and co-lipids. One or more additional physiologically acceptable substances may be included in the pharmaceutical formulation of the invention to stabilize the formulation for storage or to facilitate successful intracellular delivery of the biologically active molecules.
Co-lipids
Co-lipids, according to the practice of the invention are useful in mixing one or more cationic amphiphiles. These co-lipids would include cholesterol, N-n-hexadecyl-N, N-dihydroxyethyl ammonium bromide (HDEAB) and the like. A preferred range of molar ratio of cationic amphiphile to co-lipid is about 1:0 to 1:2.5. As such, it is within the art to vary the said range to a considerably wide extent.
Biologically active molecules that can be administered intracellularly in therapeutic amounts using the amphiphilic compounds of the invention include:
a) Ribosomal RNA
b) Antisense polynucleotide of RNA or DNA
c) Polynucleotide of genomic DNA, cDNA or mRNA that encodes for a therapeutically important protein.
The cationic amphiphiles of the invention may be blended such that one or more representatives thereof may be used in a combination to facilitate entry of the said biologically active molecules into cells/tissues.
In a further embodiment, the amphiphiles may be used either in pure form or in a combination with other lipids or amphiphiles such as helper lipids which may include phospholipids namely, cholesterol, phosphatidylethanolamine, phosphatidylglycerol etc.
In a further embodiment, the said therapeutic formulation may be stored at 0xc2x0 C. 4xc2x0 C. until complexed with the nucleic acid. Agents that prevent bacterial growth and increase the shelf life may be included along with agents that stabilize the preparation e.g., low concentrations of glycerol. It is specifically warned that freezing and thawing cycles could cause loss in efficiency of the formulation.
In another embodiment, the formulation of amphiphiles and nucleic acid may be administered intravenously besides other routes such as intramuscular and intra peritonial. Further, the said formulation of amphiphiles may be administered to cells at a ratio of 0.1-0.5 microgram of DNA to 50,000 cells in an in virto system. The amount of amphiphile could be varied from a charge ratio of 0.1 to 10, considering one positive charge for one amphiphile molecule, to one charge of nucleotide base.
The plasmid used is a construct of an Rous Sarcoma virus promoter linked to a reporter gene xcex2-galactosidase as given in the method of Banos et al (Developmental Biology 127, 209-219 (1988)). The plasmid could be of any construction and the example given is merely to demonstrate the efficiency of the amphiphilic formulation. Similar examples of plasmid include PGL-2 and PGL-3 of Promega and others.
The invention further provides a process for the preparation of the said formulation, comprising the steps of: preparing a dispersion of a cationic amphiphile disclosed in the present invention; contacting said dispersion with a biologically active molecule to form a complex between said amphiphiles and said molecule and contacting cells with said complex thereby facilitating transfer of said biologically active molecules into the cells.
In an embodiment, the invention provides a method for the treatment of cystic fibrosis and related diseases comprising the steps of administering therapeutically effective amount of the said formulation to a subject in need thereof.
In another embodiment, the said formulation may be administered through intravenous, intramuscular and intraperitonial routes.